The high incidence of tuberculosis worldwide is due to a number of factors, including the high frequency of co-infection in AIDS patients, continued emergence of drug resistance, incomplete sterilizing activity of therapeutic regimen and the ability of M. tuberculosis (Mtb) to persist in the host indefinitely. The increasing incidence of extrapulmonary tuberculosis is a grave public health concern because of its higher mortality rates and the need for extended therapy. High HIV-tuberculosis co-infection rates are a likely cause of the increasing incidence of extrapulmonary tuberculosis, since dissemination occurs in >50% of AIDS patients, but only 5-20% in HIV-negative individuals. Miliary tuberculosis, a severe form of tuberculosis occurring in approximately 8% of the extrapulmonary cases, has a much higher rate of mortality. These observations suggest that dissemination is a key factor in the severity of tuberculosis. Since lesions that lead to reactivation are secondary lesions, dissemination also plays an important role in establishment of latent infections, which affect nearly one-third of the world's population. Despite the importance of dissemination in the severity of disease and establishment of latency, the molecular mechanisms involved are poorly understood. In order to better understand how tuberculosis disseminates and whether these mechanisms result in greater disease severity and/or contribute to latency, we plan to identify and characterize the mycobacterial factors involved in both processes. There are four alternative hypotheses regarding how dissemination might occur in tuberculosis: 1) transit through the epithelium within migrating macrophages/dendritic cells, 2) direct invasion of the epithelium, 3) cytopathic and inflammatory response in epithelium leading to permeability, and 4) encroachment of granulomas into vessels allowing release of bacteria/infected cells. Currently, only a single Mtb gene, hbhA, has been identified that affects dissemination. Our own work and that of others indicate mycobacteria have numerous genes that affect the efficiency of macrophage and epithelial cell infection. It has also been shown that growth of Mtb and other mycobacterial species within epithelial cells and phagocytic cells enhances their ability to infect macrophages. The presence of numerous regulated and constitutive mechanisms of host cell infection suggest there is a spectrum of mycobacterial genes involved in tissue invasion and dissemination that remain to be identified. Our specific aims are to: 1) identify the Mtb genes involved in hematogenous spread from the lung, 2) detailed comparison of dissemination in mice and guinea pigs, and 3) dissect the molecular mechanisms by which the mel2 locus affects dissemination and persistence. We will take advantage of our interdisciplinary team's experience in mycobacterial molecular genetics, tissue culture models and animal models to provide insight into the molecular basis of dissemination and persistence, which are critical stages of tuberculosis pathogenesis. PUBLIC HEALTH RELEVANCE: Tuberculosis remains one of the most important causes of morbidity and mortality in humans worldwide, infecting nearly one-third of the world's population and causing more than two million deaths each year. Latent infections are responsible for the majority of those individuals infected and mortality is higher in extrapulmonary infections that arise from dissemination of the bacteria to other tissues. The current proposal is focused on understanding how dissemination occurs and its relationship to latency so that we can better treat, prevent and diagnose tuberculosis.